Brain Advance Access originally published online on October 17, 2005
Brain 2006 129(1):212-223; doi:10.1093/brain/awh655
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Modulating CCR2 and CCL2 at the blood–brain barrier: relevance for multiple sclerosis pathogenesis
1 Department of Neurosciences, The Lerner Research Institute, Cleveland Clinic Foundation, 2 The Mellen Center for Multiple Sclerosis Treatment and Research, Department of Neurology, Cleveland Clinic Foundation, 3 Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH, 4 Millenium Pharmaceuticals, Inc, Cambridge, MA, 5 Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, MD, 6 Department of Neurology, Newcastle General Hospital, Newcastle upon Tyne, UK, 7 Department of Internal Medicine, Nephrology Section, University of Regensburg, Regensburg, Germany and 8 Netherlands Brain Bank, Amsterdam, The Netherlands 9 Present address: Abbott Bioresearch Center, Worcester, MA, USA
Correspondence to: Richard M. Ransohoff, Neuroinflammation Research Center, Department of Neurosciences, Mail Code NC30, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA E-mail: ransohr{at}ccf.org
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Chemokines and chemokine receptors play a key role in the transmigration of leucocytes across the blood–brain barrier (BBB). CCR2 is the major receptor for CCL2, a potent monocyte and T cell chemoattractant. CCR2 and CCL2 have been consistently associated with a pathogenic role in experimental autoimmune encephalomyelitis, using knockout and transgenic mice, neutralizing antibodies, peptide antagonists and DNA vaccination. However, the significance of CCL2 and CCR2 in multiple sclerosis is enigmatic, because CCL2 levels are consistently decreased in the CSF of patients with this disease and other chronic neuroinflammatory conditions, despite abundant expression within lesional multiple sclerosis tissues. This study used an in vitro BBB model to test the hypothesis that CCL2 is removed from the extracellular fluid by CCR2-positive migrating cells as they cross the BBB, resulting in decreased CSF CCL2 levels. We showed that CCR2-positive T cells and monocytes migrated selectively across the in vitro BBB, and that CCL2 on the abluminal (tissue) side was consumed by migrating T cells and monocytes. Next, we used a new anti-CCR2 antibody to show that CCR2-positive mononuclear inflammatory cells could be readily detected in appropriate positive control tissues, but that CCR2+ cells were very infrequently found in multiple sclerosis lesions. We then showed that CCR2 receptor density on T cells and monocytes was specifically downregulated upon in vitro BBB transmigration in response to CCL2, but not irrelevant chemokines. These findings document a novel strategy for analysing chemokine receptor function in inflammatory CNS disease, and support the hypothesis that CCL2 is consumed by migrating inflammatory cells, which downregulate CCR2, as they cross the BBB.
Key Words: multiple sclerosis; chemokines; chemokine receptors; CCR2; CCL2/MCP-1
Abbreviations: IVBBB = in vitro blood–brain barrier; MCP = monocyte chemoattractant protein; MFI = mean fluorescence intensity; PBMC = peripheral blood mononuclear cells; THBMEC = transformed human brain microvascular endothelial cell; WNE = West Nile encephalomyelitis
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Received July 26, 2005. Revised September 7, 2005. Accepted September 12, 2005.
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In chronic inflammatory CNS disorders, the haematogenous infiltrate consists of mononuclear cells, primarily T cells, monocytes and macrophages. As with all leucocyte extravasation events, key players in the transmigration of T cells and monocytes across the blood–brain barrier (BBB) include selectins, integrins, cell adhesion molecules, chemokines and matrix metalloproteases. Chemokines interact with G-protein coupled high-affinity receptors and govern the migration of leucocytes during both physiological and pathological processes (Rot and von Andrian, 2004
). In addition to their roles in transmigration at the BBB, chemokines also mediate migration of T cells, monocytes and microglia within CNS parenchyma (Tran and Miller, 2003). The terminology for chemokines and their receptors was recently systematized (Zlotnik and Yoshie, 2000; Bacon et al., 2002) and is regularly updated on a dedicated website (http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html).
CCR2 is a CC chemokine receptor that responds predominantly to monocyte chemoattractant protein-1 (MCP-1; CCL2 in the systematic nomenclature), one of the first CC chemokines to be characterized and associated with pathological inflammation. In humans, CCR2 is expressed by virtually all monocytes and 
15% of CD4+ T cells in the circulation, where CCR2+ T cells also express markers of chronic activation such as CD26 (Kivisakk et al., 2002). CCL2 is present in a large variety of clinically important inflammatory and infectious disorders (Mahad and Ransohoff, 2003). Genetic evidence supporting a direct role for CCL2 in human inflammatory diseases was recently reported: renal biopsy tissue from patients with systemic lupus erythematosus nephritis contained greater number of CD68+ cells in CCL2 high secretors (–2578G allele) compared to low secretors (Gonzalez et al., 2002). Further, carriers of the CCL2 –2578G high secretor allele had elevated levels of CCL2 in CSF (Letendre et al., 2004), and were much more prone to developing dementia following HIV infection (Gonzalez et al., 2002). Interestingly, increased CCL2 in CSF preceded and predicted dementia in simian immunodeficiency virus encephalitis, a non-human primate model of HIV-associated dementia (Zink et al., 2005).
Unexpectedly, when we surveyed CSF chemokines in patients with multiple sclerosis, we found significantly less CCL2, as compared with those patients having non-inflammatory neurological disorders (NINDs). This finding was selective for CCL2, as other chemokines were either elevated (CXCL10) or equal in comparison to multiple sclerosis and NIND CSF. Subsequently, numerous additional independent studies reported a consistent, significant decrease in CSF CCL2 levels in patients with multiple sclerosis, compared to controls, with further decreases in patients during clinically or radiographically active disease (Sorensen et al., 1999; Franciotta et al., 2001; Sindern et al., 2001; Sorensen et al., 2001, 2004; Mahad et al., 2002; Scarpini et al., 2002; Bartosik-Psujek and Stelmasiak, 2005). Typically, NIND CSF contains 2–300 pg/ml of CCL2, with a consistent 50% decrease in multiple sclerosis CSF. In Japanese patients, CSF CCL2 levels were elevated in relapsing neuromyelitis optica while being reduced in multiple sclerosis (Narikawa et al., 2004). CCL2 was also reduced in CSF from patients with several other chronic neuroinflammatory conditions with prominent leucocyte infiltrates, including HTLV 1-associated myelopathy, Behçet's disease and Vogt–Harada–Koyanagi syndrome (Saruhan-Direskeneli et al., 2003; Miyazawa et al., 2005; Narikawa et al., 2005). By contrast, chronic conditions such as amyotrophic lateral sclerosis, that lack abundant leucocyte infiltration, showed elevated CSF CCL2 levels, or showed no difference from controls (Baron et al., 2005). Acute neuroinflammatory conditions including trauma, bacterial and viral meningitis, as well as HIV-1-associated encephalopathy, featured CCL2 CSF levels which were extraordinarily elevated (Conant et al., 1998).
What factors could account for reduced CCL2 in multiple sclerosis CSF? This finding must reflect either decreased production or increased consumption. Accordingly, it is conceivable that CCL2 production might be suppressed in the highly Th1-biased environment of the active multiple sclerosis lesion, since this chemokine is associated in some models with Th2 polarization (Gu et al., 2000). Arguing against this hypothesis, examination of tissue sections from multiple sclerosis cases shows abundant CCL2 immunoreactivity in active and chronic active lesions (McManus et al., 1998; Simpson et al., 1998; Van Der Voorn et al., 1999). In the current study, we addressed the possibility that increased CCR2-mediated consumption, which is known from in vitro studies to be avid (Tylaska et al., 2002), might account for altered CCL2 levels in the CSF of multiple sclerosis patients. In this regard, little is known about the expression of CCR2 in human CNS tissues, mainly because of limited immunohistochemical reagents, although one study reported CCR2 immunoreactivity on infiltrating mononuclear cells in multiple sclerosis lesions using monoclonal anti-CCR2 antibodies (Simpson et al., 2000). Data about CCR2 expression in human inflammatory disorders are relevant in view of the recent interest in blockade of CCR2 for clinical application (Daly and Rollins, 2003).
A recent publication listed distinctive phenotypes for CCR2–/– mice in 21 disease models that included atherosclerosis, arthritis, transplantation, pulmonary fibrosis, neuropathic pain and glomerulonephritis (Quinones et al., 2004). Therefore, CCR2 is the best-characterized and most widely implicated chemokine receptor in models of human disease. In vivo, the CCL2/CCR2 interaction mediates the recruitment of CCR2-bearing leucocytes into the CNS in a non-redundant manner, as shown in a number of genetic animal models as well as through the use of CCL2 neutralizing agents. In murine EAE, CCL2 mRNA expression in the brain and spinal cord was upregulated in close association with EAE attacks (Ransohoff et al., 1993; Glabinski et al., 1997). Neutralization with anti-CCL2 antibodies, or through DNA vaccination before immunization, protected mice against EAE (Kennedy et al., 1998; Youssef et al., 1999). In CCL2 knockout mice with EAE, macrophage recruitment was impaired and the clinical disease was much less severe with diminished relapses compared to wild-type mice (Huang et al., 2001). Two independent attempts to induce EAE in CCR2 knockout mice were unsuccessful (Fife et al., 2000; Izikson et al., 2000). Using rather vigorous methods of EAE induction, one study described a neuroinflammatory reaction, with a predominant neutrophil infiltrate in the CNS in CCR2 knockout mice (Gaupp et al., 2003).
CCL2 and CCR2 also play essential roles in the recruitment of monocytes into lesions of spinal cord contusion in rodents (Ma et al., 2002), demonstrating a more general role for this chemoattractant system in neuroinflammation. In the intact CNS, in situ hybridizations show that CCL2 is produced in highly restricted fashion either by neurons or parenchymal CNS astrocytes (Glabinski et al., 1997; Ma et al., 2002), giving rise to the hypothesis that the chemokine, produced abluminally, is transcytosed by endothelial cells before presentation to circulating leucocytes (Rot and von Andrian, 2004; Dzenko et al., 2005).
Here we provide data to address the unexpected findings of reduced CCL2 in multiple sclerosis CSF. We used an in vitro blood–brain barrier (IVBBB) model, composed of transformed human brain microvascular endothelial cells (THBMEC) in transwell culture, to examine the hypothesis that CCL2 is removed from the CNS extracellular fluid by CCR2-positive migrating cells, leading to decreased CCL2 levels. In the IVBBB model, as in formation of multiple sclerosis lesions, monocytes (100% of which were CCR2-positive) migrated much more efficiently than T cells. Only CCR2-positive lymphocytes migrated across the BBB in vitro, leaving CCR2-negative lymphocytes in the non-migrated population. Further, CCL2 on the abluminal side of the THBMEC monolayer was consumed by migrating peripheral blood mononuclear cells (PBMC). We used a novel anti-CCR2 antibody to show that very few CCR2-positive mononuclear inflammatory cells were identified in multiple sclerosis lesions, suggesting that the receptor had been down-regulated by ligand-induced migration into lesions. Consistent with this proposal, we found that CCR2 was downregulated on both monocytes and T cells when CCL2 was used as the chemoattractant, but the receptor was not downregulated when cells migrated in response to an irrelevant chemokine.
Overall, our data supported the hypothesis that CCL2 is consumed by CCR2+ migrating cells, providing a potential explanation for the consistent finding of decreased CSF CCL2 in several neuroinflammatory disorders, including multiple sclerosis. Our findings also supported the possibility that CCR2 is downregulated on cells which migrate in response to CCL2, but not in response to irrelevant chemokines. We demonstrate in this study a novel approach to evaluating the significance of chemokine levels in body fluids and chemokine receptors on cells found in tissue sections. This approach is clinically relevant, given the availability of small molecule compounds and monoclonal antibodies that block CCR2, and can be used as potential treatments for human inflammatory disorders.
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Leucocyte preparation for migration assays
PBMC were isolated from fresh whole heparinized blood from healthy volunteers by density centrifugation using Lymphocyte Separation Medium (Mediatech, Herndon, VA). After washing, PBMC were centrifuged at 300 g to remove platelets. The final cell population was resuspended at 107 cells/ml in RPMI 1640 without phenol red + 1% BSA [transendothelial migration (TEM) buffer] for transmigration assays. When indicated, 107 PBMC/ml were pre-treated with 100 ng/ml pertussis toxin (Sigma, St Louis, MO) for 1 h at 37°C and washed extensively prior to resuspending at 107 cells/ml in TEM buffer.
Transmigration assays
Transmigration assays were performed as described previously (Callahan et al., 2004). Transformed human brain microvessel endothelial cells (THBMEC) were used in transmigration assays at passages 15–25 (Stins et al., 1997, 1999, 2001; Callahan et al., 2004). BBB characteristics of the THBMEC cultures were routinely tested by assessing transendothelial electrical resistance (TEER) using an EVOM voltohmmeter (World Precision Instruments, Sarasota, FL) and assays were performed when TEER was >80 ohm-cm2. Expression of tight junction proteins occludin and ZO-1 was previously confirmed by immunocytochemistry and western blotting (Callahan et al., 2004). Transmigration assays were performed by first growing THBMEC to confluence in transwell inserts, which were transferred to fresh wells containing 600 µl TEM buffer. CCL2, CCL5 or CCL3 (R&D Systems, Minneapolis, MN) were included in the lower chamber at varying concentrations when indicated. 106 PBMC in 100 µl TEM buffer were added to the top chamber and allowed to transmigrate at 37°C in a humid atmosphere of 5% CO2 for 3 h. In indicated experiments, PBMC were fluorescently labelled with calcein-AM (Molecular Probes, Eugene, OR) according to manufacturer's directions and transmigrated PBMC were counted in samples from the bottom chamber by measuring fluorescence at 530 nm using a SPECTRAmax GEMINIXS microplate spectroflurometer (Molecular Devices Corporation, Sunnyvale, CA). Fluorescence values of migrated PBMC were compared to a standard curve of known cell numbers for quantitation. When PBMC were subsequently analysed by flow cytometry, the calcein-AM labelling was omitted.
For ELISA assays to determine CCL2 consumption by migrating cells, samples were obtained from 3 h migration assays that included 10 ng/mL CCL2 in the lower chamber and were performed in the absence or presence of PBMC in the top chamber (see above). Where indicated, PBMC were fixed with paraformaldehyde (1.5%) for 15 min at room temperature, followed by washing twice, before addition to the top chamber, to provide negative control data points. Supernatants and cells, when applicable, were collected from the top and bottom chambers, centrifuged, and the supernatants carefully transferred to fresh tubes and stored at –20°C until further analysis.
Antibody staining and flow cytometry
Flow cytometric analysis of input, non-migrated and migrated PBMC was performed in parallel with input cells stained at 0 h and non-migrated and migrated cells stained after 3 h. Non-migrated PBMC were recovered by carefully removing the fluid from the top of the insert. Migrated PBMC were recovered by gently titurating media and PBMC from the bottom chamber. At least two wells were pooled for flow cytometric analysis of migrated PBMC of which PBMC were blocked with 0.2 mg/ml normal goat IgG (Caltag Laboratories, Burlingame, CA) and incubated with anti-CCR2 antibody (Doc-3; generated by M. Mack) for 15 min on ice. After washing twice with PBS + 2% heat-inactivated FCS + 0.1% sodium azide (FACS buffer), PBMC were stained with 1 : 125 anti-mouse IgG1-PE (Southern Biotechnology Associates, Birmingham, AL) for 15 min on ice, followed again by two washes. The third staining step included staining with anti-CD14-FITC (clone SK3; BD Biosciences, San Jose, CA) and CD3-PerCP (clone SK7; BD Biosciences) for 15 min on ice, after which PBMC were washed twice with FACS buffer, fixed in 1% paraformaldehyde, and data acquired using an LSR flow cytometer (BD Biosciences). Analysis was performed using WinList software (Verity Software House, Topsham, ME). Lymphocytes and monocytes were gated according to forward and side scatter, as well as CD14 and CD3 staining profiles, and analysed against isotype matched controls.
CCL2 ELISA
Samples for ELISA were collected as described above (see Transmigration assays). ELISA for human CCL2 was performed according to manufacturer's directions using the DuoSet ELISA kit (R&D Systems). Briefly, flat bottom 96 well plates were coated overnight with anti-CCL2 capture antibody at 4°C. Plates were washed, blocked with blocking buffer provided in the kit and incubated with 100 µl samples, in triplicate, for 2 h at RT. After washing, plates were incubated with anti-CCL2 detection antibody conjugated to biotin for 2 h, followed by a 20 min incubation with streptavidin–horseradish peroxidase. Substrate was added for a final 20 min incubation, after which absorbance measurements were taken at 450 nm for protein concentration determination, and 540 for background measurements using a SpectraMax M2 microplate reader (Molecular Devices, Sunnydale, CA). Background measurements (taken at 540 nm) were subtracted from protein measurements (taken at 450 nm) and CCL2 concentrations (pg/ml) were determined against a standard curve using manufacturer's instructions.
Autopsy and surgical resection material from CNS pathologies
Autopsy material from multiple sclerosis cases was collected at the Netherlands Brain Bank and the Cleveland Clinic Foundation according to their respective rapid autopsy protocols (Ravid and Swaab, 1993; Chang et al., 2002). Autopsy and surgical resection tissue from a variety of other neurological disorders was collected at the Cleveland Clinic Foundation. All cases underwent routine gross and histopathological evaluation. A total of 13 tissue sections of 9 individual cases were included in the analysis of CCR2 expression (Table 1). Under guidelines established by the National Institutes of Health (USA), the study of the autopsy and surgical resection material was exempt from review, as determined by the Institutional Review Board (IRB) of the Cleveland Clinic Foundation.
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Immunofluorescence histochemistry
Immunofluorescence histochemistry was performed on 35 µm thick free-floating sections. The tissue was fixed in 4% paraformaldehyde for 48 h, protected in 70% glycerol overnight and then placed on the stage of a sliding microtome for cutting. Free-floating sections were rinsed in PBS four times for 5 min each, microwaved twice for 5 min each in 10 mM citrate buffer (pH 6.0), incubated in 3% H2O2 and 10% Triton X-100 for 30 min. The non-specific binding was blocked with 10% human serum + 0.1% Triton X-100. Table 2 indicates the primary antibodies that were used in free-floating sections, which were incubated for 6 days at room temperature (RT). Sections were imaged using a Leica DMR microscope (Leica Wetzlar, Heidelberg, Germany) microscope and an Optronix Magnafire digital camera system and analysed using Image Pro® Plus (Media Cybernetics, Silver Springs, MD).
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In a comprehensive approach to detection of CCR2 immunoreactivity, 5 µm thick fresh frozen sections, fixed paraffin embedded sections (5 µm) and free-floating sections were all used. The distribution of immunoreactivity of all anti-CCR2 antibodies was screened using immunoperoxidase histochemistry and brightfield microscopy. The frozen sections were thawed at RT, fixed in ice-cold acetone for 7 min and washed in PBS + 0.1% Triton X-100 (Sigma) three times prior to blocking with 10% human serum (Sigma) for 1 h at RT. Following the incubation of primary antibodies, sections were immunostained using avidin–biotin complex procedure and diaminobenzidine, as described previously (Sorensen et al., 1999
). The endogenous peroxidases were inactivated using 3% H2O2 for 10 min at RT. The frozen sections were counterstained using haematoxylin (Fisher Scientific, Pittsburgh, PA). The specificity of anti-CCR2 (1D9) antibody was determined by pre-incubating the anti-CCR2 (1D9) antibody with the peptide (MLSTSRSRFIRNTNESGEEV, generated by Invitrogen Life Technologies, Carlsbad, CA), identical to what was used to generate the mouse monoclonal anti-CCR2 (1D9), at a concentration of 60 mg/ml (diluted in PBS) at RT for 2 h. To confirm specificity of the peptide, it was also pre-incubated (60 mg/ml) with anti-LCA antibody (DAKO) in parallel experiments.
For the co-localization of CCR2 (1D9) with CD3, mononuclear phagocyte markers (CD68, MRP-14, and iba-1), or endothelial cell markers (von Willebrand factor; vWF), the appropriate pairs of primary antibodies (Table 2) were incubated simultaneously at 4°C for 6 days. Following the primary antibody incubation, free-floating sections were washed in PBS + 0.1% Triton X-100 three times prior to the addition of biotinylated anti-mouse IgG2A (Southern Biotechnology) at 1 : 250 dilution (in PBS + 0.1% Triton X-100) together with either Texas Red conjugated anti-rabbit IgG (Southern Biotechnology) at 1 : 250 dilution for CD3 and iba-1, or Texas Red conjugated anti-mouse IgM (Southern Biotechnology) at 1 : 250 dilution for CD14 or Texas Red conjugated anti-mouse IgG1 (Southern Biotechnology) at 1 : 250 dilution for CD68 and MRP-14. Neutralite avidin-FITC (Southern Biotechnology) at 1 : 100 dilution (in PBS) was used to detect the biotinylated anti-mouse IgG2A (Southern Biotechnology). Double labelled free-floating sections were mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA). The potential cross-reactivity of secondary antibodies was tested by omitting one primary antibody.
Confocal microscopy
Double labelled sections were analysed on a Leica Aristoplan laser scanning microscope (Leitz Wetzlar, Heidelberg, Germany). Individual confocal optical sections represented 0.5 µm axial resolution. The images presented here consist of 12–20 optical sections combined to form a ‘through-focus’ image. Fluorescence was collected individually in the green (fluorescein) and red (Texas Red) channels to eliminate ‘bleed-through’ from either channel.
CCL2 binding assay
Frozen 5 µm sections from multiple sclerosis cases were thawed at RT for 1 h and hydrated in PBS for another h at RT. A biotinylated recombinant human CCL2/MCP-1 (biot-rhMCP-1, Fluorokine kit, R&D Systems) at 1 : 4 dilution (3 µg/ml in PBS) was added to the sections and incubated for 3 h at RT. Excess biot-rhMCP-1 was removed by tipping the slides and avidin-FITC was added to the slides and incubated for 1 h at RT. Unbound biot-rhMCP-1 and avidin-FITC were removed by washing in PBS three times. Sections were mounted in Vectashield with DAPI. In order to demonstrate whether anti-CCR2 antibody (1D9) interferes with the binding of rhMCP-1, frozen hydrated sections were incubated with anti-CCR2 antibody (1D9) at 1 : 50 dilution for 1 h at RT prior to the addition of biot-rhMCP-1 in parallel experiments. Antibodies that do not bind to CCR2 (CXCR3 and GFAP from BD Pharmingen, San Diego, CA and DAKO, respectively) as well as non-biotinylated CCL3/MIP-1
, non-biotinylated rhMCP-1 and avidin-FITC without biot-rhMCP-1 were used as controls.
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The migration of CCR2+ across the IVBBB cells is enhanced by CCL2 in a gradient and concentration dependent manner, and is pertussis-toxin sensitive
We characterized the relative proportions of PBMC that were CD14+ monocytes and CD3+ T cells in input, non-migrated and migrated populations in the IVBBB model. 10–15% of PBMCs migrated under baseline condition without exogenously added chemokine in a 3 h migration assay (defined as 100% basal migration; Fig. 1). Flow cytometric analysis showed that >80% of migrated cells were CD14+ monocytes and 5% were CD3+ T cells, contrasting sharply with the input population (65% T cells and 16% monocytes; Table 3). Monocytes were substantially depleted from the non-migrated population consistent with the preferential migration of input monocytes across the IVBBB.
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In order to compare the ability of PBMC to migrate under baseline and chemokine-driven conditions, CCL2 was included in the lower chamber of the IVBBB at varying concentrations in transmigration assays. Dose–response experiments demonstrated the characteristic bell-shaped curve observed in such assays (Fig. 1). CCL2-mediated migration was inhibited by pertussis toxin, consistent with G
i signalling (Murphy, 1994) (data not shown). Installation of equal concentrations of CCL2 in the top and bottom chambers reduced the migration of input PBMC to baseline levels, consistent with a chemotactic rather than chemokinetic effect (data not shown). Addition of neutralizing anti-CCR2 antibodies to the upper chamber suppressed CCL2 driven migration of PBMCs across the IVBBB (data not shown), confirming the functional importance of CCL2 signalling to CCR2 in this system. CCL3 (MIP-1), which signals to CCR1 and CCR5 but not CCR2, augmented PBMC migration across the IVBBB, with a similar dose dependency (data not shown), demonstrating that chemokine-induced migration is not limited to CCL2 in this model. At the optimal concentration of 25 ng/ml, CCL2 led to a significant increase in the number of migrated PBMC from 150 000 at baseline, up to 350 000 in a 3 h assay (Fig. 1). Kinetic assays using 25 ng/ml of CCL2 showed that migration was significantly enhanced after 1 h, and that the effect was maintained at least through 5 h. The relative proportions of migrating monocytes and T cells were not significantly altered by CCL2, indicating that the chemokine effect was exerted equally towards monocytes and T cells. In particular, kinetic analysis at the optimal CCL2 concentration of 25 ng/ml showed that monocytes responded rapidly (at 1 h; 200% of baseline), while CD3+ T cell responses to CCL2 were observed at 3 h (170%) and 5 h (130%). This result implicated CCR2 in the response of both monocytes and T cells to CCL2, in this system.
We evaluated CCR2 expression on input, non-migrated and migrated PBMC, gating either on CD14+ monocytes or CD3+ lymphocytes. More than 90% of CD14+ monocytes were CCR2+ in all populations (data not shown). Comparison of migrated and non-migrated PBMC showed a significantly greater percentage of CCR2+, CD3+ lymphocytes in the migrated population in response to CCL2, CCL3 and CCL5, or without exogenous chemokine, consistent with greater migratory capacity of chronically activated CCR2+ T cells (Table 4). Interestingly, CD3+ T cells increased their migration more robustly in response to CCL2 than to CCL3 or CCL5, which have been considered as relatively selective T cell chemoattractants. CXCL10 did not increase migration in this system at any concentration, consistent with our previous finding that CXCR3, the CXCL10 receptor, was not implicated in lymphocyte transmigration in this IVBBB system (Callahan et al., 2004). These results indicated that CCL2, CCL3 and CCL5 attracted both T cells and monocytes across the IVBBB, with approximately equal efficiency, in 3 h transmigration assays.
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CCR2 receptor density is down-regulated on migrating monocytes and T cells during CCL2-induced but not CCL3-induced transmigration
To determine whether CCR2 expression was modulated on PBMC upon baseline or chemokine-driven migration, we evaluated CCR2 receptor density or input, non-migrated and migrated populations of CD3+ T cells and CD14+ monocytes by measurement of mean fluorescence intensity (MFI) using flow cytometry. Strikingly, CCR2 MFI was significantly reduced on T cells (Fig. 2A) and monocytes (Fig. 2B) that had migrated in response to CCL2 compared either to input cells or the migrated populations from baseline, CCL3-, or CCL5-driven migration assays. All three chemokines tested in this system augmented migration by both T cells and monocytes. However, CCR2 MFI on both T cells and monocytes was significantly reduced only in CCL2-driven migration assays. This result demonstrated that T cell expression of CCR2 was downregulated selectively during transmigration in response to CCL2, but not by migration towards CCL3 or CCL5. Migrated T cells were markedly enriched for CCR2 positivity, compared to input cells, regardless of the stimulus to migration. However, after migrating towards a CCL2 gradient, T cells expressed CCR2 at greatly reduced density per cell (Table 4; Fig. 2A). This result suggested that CCR2 was downregulated by engaging CCL2, which was ‘presented’ on the luminal surface of the brain microvascular endothelial cells (Hardy et al., 2004
). CCR2 was not downregulated simply by T cell/endothelial contact since non-migrated populations exhibited MFI consistent with input populations after CCL2-driven migration (data not shown). To ensure that the decreased MFI was not a result of steric interactions between CCL2 and CCR2 antibody, we showed unaltered CCR2 MFI despite including exogenous CCL2 in the staining reaction mixture (15 min at 4°C; data not shown). Longer exposure of PBMC to CCL2 in solution at room temperature downregulated CCR2, as previously shown by others (data not shown).
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CCL2 is consumed by migrating PBMCs during transmigration across the IVBBB
During chemokine signalling, receptor–ligand complexes are internalized (Pelchen-Matthews et al., 1999
). We determined whether migrating PBMC depleted CCL2 through receptor-mediated internalization, given the reduced CSF CCL2 levels in multiple sclerosis patients. We evaluated CCL2 levels in the top and bottom chambers by ELISA following CCL2-driven migration. The addition of CCL2 to the bottom (abluminal) chamber led to an increase in the CCL2 levels in the top (luminal) chamber within 1 h, likely due to transcytosis of CCL2 by the THBMEC (Dzenko et al., 2005) in the IVBBB (data not shown). When migration of PBMCs had proceeded for 3 h, CCL2 levels in the top and the bottom chambers were compared to those obtained in the absence of PBMC (Table 5). Removal of CCL2 per 105 cells was much greater in the bottom chamber than in the top chamber (mean 298.9 pg CCL2/100 000 PBMCbottom compared to 5.9 pg CCL2/100 000 PBMCtop), during migration by T cells and monocytes. Paraformaldehyde-fixed PBMCs did not affect CCL2 levels (data not shown), indicating that it was not sufficient for CCL2 to bind to surface expressed CCR2, but rather that active internalization was required, to reduce CCL2 concentrations.
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CCR2 is expressed sparsely on T cells and not at all on monocyte lineage cells in multiple sclerosis lesions
Data concerning CCR2 immunoreactivity in CNS tissue has been limited due to the lack of available reagents. The current study used 1D9 anti-CCR2 antibodies to analyse CNS tissues (Fig. 3). CCR2+ cells were detected only infrequently in multiple sclerosis lesions, comprising <5% of T cells, using 10 sections from seven different autopsy cases (Table 1). Where present in multiple sclerosis tissues, 1D9 immunoreactivity identified a small proportion of perivascular cuff T cells in lesions (Fig. 3D–F). CCR2 immunoreactivity was not detected on CD14, CD16, CD68 or iba-1 immunoreactive cells in the tissue sections (data not shown). In contrast, the majority of T cells in spinal cord tissue from West Nile encephalomyelitis (WNE) cases (n = 2) expressed CCR2 (Fig. 3A–C). Very few non-T cells showed CCR2 immunoreactivity (Fig. 3C, arrowheads). In WNE tissues, these cells were identified as activated MRP-14+ monocytes, but CCR2+ cells represented a tiny fraction of all monocytes (data not shown). Given our findings in WNE spinal cord sections, we also analysed spinal cord sections from multiple sclerosis cases with 1D9. We failed to detect significantly more CCR2-immunoreactive cells in these spinal cord sections as compared with multiple sclerosis brain sections.
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Confirming CCR2 immunoreactivity in multiple sclerosis tissue sections
Because 1D9 immunoreactivity has not been characterized in CNS tissues, we performed extensive control experiments. 1D9/CCR2 immunoreactivity was eliminated by pre-incubation with an excess of the immunizing peptide (data not shown). Further support for the authenticity of 1D9/CCR2 immunoreactivity in multiple sclerosis tissue was obtained using a CCL2 binding assay. Binding of biotinylated-CCL2 to membranes of small round cells (identified using DAPI; Fig. 4A, inset), was consistent with the distribution of CCR2 immunoreactivity (Fig. 4). Furthermore, pre-incubating multiple sclerosis tissue with anti-CCR2 (1D9) antibodies blocked the binding of biotinylated-CCL2, confirming that CCR2 was the relevant binding site (Fig. 4B). The pre-incubation of multiple sclerosis tissue with irrelevant antibodies (CD68, CXCR3 and GFAP) did not affect CCL2 binding (Fig. 4C and D and data not shown). The incubation of sections of multiple sclerosis tissue with avidin-FITC alone, without biotinylated CCR2, produced only background staining (data not shown). Excess unlabelled CCL2 significantly decreased biotinylated-CCL2 binding (Fig. 4E). Incubation with unlabelled CCL3, either before or during the binding reaction, failed to alter biotinylated-CCL2 binding to multiple sclerosis tissue sections (Fig. 4F). Other anti-CCR2 antibodies failed to show consistent cellular immunoreactivity in any CNS tissue sections, despite the use of a variety of staining techniques (Table 2).
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Discussion |
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In the current study, the following findings were made: monocytes (all of which were CCR2+) and CCR2+ T cells migrated across the IVBBB with enhanced efficiency compared to CCR2-negative cells; CCL2 augmented both monocyte and T cell migration but was removed from the medium during the process, while CCR2 was downregulated on migrating cells; and CCR2 immunoreactivity was not frequently detected in inflamed multiple sclerosis lesions. These data were interpreted in the context of prior reports showing reduced CSF CCL2 concentrations during this disease. Taken together, the data suggest a potential role for CCL2 and CCR2 in leucocyte accumulation in multiple sclerosis lesions.
A number of chemokines and their receptors have been considered as attractive targets for immunomodulation of inflammatory CNS and systemic disorders, such as multiple sclerosis. Currently available immunomodulatory agents are effective in reducing the rate of relapses as well as the formation of new lesions as determined by gadolinium enhanced MRI. However, not all patients respond to currently available agents and multiple agents that reduce inflammation may be more beneficial than a single agent. Chemokines and chemokine receptors are potential therapeutic targets to curtail the inflammatory infiltrate in multiple sclerosis (Proudfoot et al., 2003; Proudfoot, 2002). Recent results indicated that leucocyte trafficking is a good therapeutic target for treating this disease (Miller et al., 2003) but that unanticipated side effects may result from blocking widely expressed leukointegrins (Berger and Koralnik, 2005). This occurrence makes it highly important to understand how the inflamed CNS recruits leucocytes from the bloodstream during multiple sclerosis.
The role of CCL2 and CCR2 in the pathogenesis of multiple sclerosis is not well understood despite the consistent and robust evidence of a pathogenic role in EAE using neutralization, knockout and transgenic models. CCL2 immunoreactivity has been readily detected in lesions (McManus et al., 1998; Van Der Voorn P et al., 1999; Simpson et al., 1998), but the same chemokine is significantly and selectively decreased in the CSF of patients (Sorensen et al., 1999). Further, decreased CSF CCL2 has only been reported in multiple sclerosis and several other neuroinflammatory states, all of which feature prominent and chronic leucocyte infiltrates. As noted above, CSF CCL2 is increased in acute neuroinflammatory disorders including viral or bacterial meningitis. The current study does not address reasons for this discrepancy between acute and chronic disorders. One consideration comes from the differing kinetics of the varied disorders: CSF CCL2 is much more likely to be at steady state in chronic than acute CNS disorders at the time of lumbar puncture. It seems likely that CCL2 production might exceed consumption during the early phase of meningitis, when CSF sampling is performed. CSF CCL2 is notably elevated in patients with HIV-associated encephalopathy, a chronic disorder. In this setting it seems plausible that robust astrogliosis enhances CCL2 production, while only sparse CCR2+ CD4+ T cells are available to consume this chemokine.
Several potential explanations have been advanced to account for reduced CSF CCL2 in multiple sclerosis. In the current report, we propose that CCR2+ cells (lymphocytes and monocytes) might migrate efficiently across the BBB and deplete CCL2 during that process. This hypothesis was supported by data obtained using the IVBBB model, which used immortalized human brain microvascular endothelial cells, to optimize experimental reproducibility, at the expense of relatively low TEER. As previously shown, T cells migrated less efficiently than monocytes, and CD14+ monocytes constituted 15% of the input population, but were enriched to 85% of the migrated cells. Nevertheless, we found that CCR2+ chronically activated T cells migrated very efficiently compared with CCR2-negative T cells, being enriched from 25–30% of the input CD3+ population to 75–80% of the migrated population. Memory T cells and un-activated monocytes both express high levels of CCR2, compared to naïve T cells and activated monocytes in the bloodstream (Carr et al., 1994). Memory T cells and monocytes are predominantly responsible for inflammatory infiltrates. Gratifyingly, this cell population profile was recapitulated in the migrated population of the IVBBB system. Overall, however, T cell migration in the IVBBB system was less efficient, so that migration of a small population of CCR2+-enriched lymphocytes (5000–10 000 cells) did not reduce the per cent CCR2+ T cells in the non-migrated lymphocyte population of 600 000–650 000 total cells (Table 4).
The CCR2+ population of migrating cells avidly consumed CCL2 from the abluminal (bottom) chamber of the IVBBB system. Migrated cells removed 50-fold more CCL2 than non-migrated cells on a per-cell basis. Prior reports using CCR2–/– and CCR2+/+ mouse splenocytes indicated that this CCL2 consumption was receptor-mediated (Tylaska et al., 2002). Our current findings were consistent with the hypothesis that reduced CSF concentrations of CCL2 are associated with its consumption by migrating cells during formation of multiple sclerosis lesions.
This study identified an enhanced capacity of CCR2+ PBMCs to migrate across an IVBBB model, raising the question of the fate of these cells, and the regulation of receptor expression in lesions. We addressed the distribution of CCR2 immunoreactivity in multiple sclerosis lesions using immunohistochemistry, with a panel of CCR2 antibodies and a variety of antigen-retrieval techniques (Table 2). We obtained consistent, reproducible CCR2 immunoreactivity using only one of six monoclonal and polyclonal antibodies (1D9) and observed scanty CCR2 expression, restricted to T cells, in lesions. The CCR2 detected by 1D9 was functional, as confirmed by CCL2 binding, which could be blocked by anti-CCR2 antibodies. Our results differed somewhat from a previously described report of CCR2 immunoreactivity in lesions, in that we observed a much more limited immunoreactivity pattern (Simpson et al., 2000). Our findings regarding sparse CCR2 immunoreactivity on mononuclear cells in lesions was not technical, as indicated by results of studying positive WNE control cases. Virtually nothing is known about CNS or CSF CCL2 levels in WNE. CCL5, CCL3, and CCL4 mRNAs were elevated in mice infected with West Nile virus (Shirato et al., 2004). The high-level expression of CCR2 on infiltrated T cells in WNE tissue is consistent with the possibility that CCL2 is not a critical determinant of leucocyte recruitment to the inflamed CNS in this disease process.
CCR2 was specifically downregulated in response to CCL2 engagement during transmigration in the IVBBB system. Ligand-induced receptor internalization is a well-documented phenomenon among chemokine receptors (Pelchen-Matthews et al., 1999). Following ligand engagement, the surface expression of chemokine receptors is dependent upon the degree of recycling of receptor to the plasma membrane. Recycling is not a uniform feature of all chemokine receptors. Recycling of CCR5, for example, is well documented, but not so for CCR1 (Elsner et al., 2000). CCR2 internalization has been proposed to regulate CCL2 levels (Tylaska et al., 2002), since CCL2 was rapidly and avidly consumed by wild-type macrophages, but not at all by CCR2–/– macrophages. Thus, utilization of CCL2 by CCR2 may, in itself, provide a feedback mechanism for controlling levels of available chemokine.
Our data support a model in which parenchymal CCL2, produced primarily by reactive astrocytes during multiple sclerosis inflammation, is consumed by CCR2+ migrating mononuclear cells as they enter the lesions. Upon ligand engagement, CCR2 is downregulated on migrating cells. Together, these processes lead to the scarcity of CCR2+ leucocytes in lesions and decline in CSF CCL2 levels. This is the first report to provide evidence for a mechanism that unifies the various descriptive observations regarding CCL2 and CCR2 in multiple sclerosis clinical-pathological material and, overall, supports the intent to examine CCR2 as a therapeutic target.
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* These authors contributed equally to this work
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Acknowledgements |
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This research was supported in part by the National Institutes of Health (NS38667 to RMR), postdoctoral fellowship FG1482 (to MKC) from the National Multiple Sclerosis Society and the Nancy Davis Center Without Walls. We acknowledge the MS Women's Committee for providing support for imaging software and a computer station for image analysis. We acknowledge Cathy Shemo and Anne Cotleur for help with flow cytometry and phlebotomy, Jerome Wujek and Bruce Trapp for iba-1 monoclonal antibody and helpful comments about the data, Judith Drazba and the LRI Confocal Core for excellent technical assistance, and members of the Ransohoff lab for advice and suggestions.
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